PROJECT SUMMARY Mammalian platelets are small anucleate blood cells specialized to continuously monitor and preserve the integrity of the cardiovascular system (hemostasis). They are produced by megakaryocytes (MKs) in the bone marrow and released into blood, where they circulate for ten days in humans and five days in mice. Platelet homeostasis, i.e. the establishment of a defined peripheral platelet count (PPC), requires that both processes - platelet production and clearance - are tightly regulated. At the same time, platelets depend on a very sensitive signaling machinery that facilitates platelet adhesion and hemostatic plug formation under shear stress. This high sensitivity, however, poses a risk for unwanted platelet activation that can lead to platelet clearance and/or thrombosis. We and others identified a critical role for the small GTPase Rap1 in platelet activation. We further demonstrated that Rap1 activity in platelets is regulated by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI, RasGRP2), and the GTPase-activating protein, Rasa3 (GAP1IP4BP). CD-GEFI senses small changes in intracellular calcium and is crucial for the rapid activation of Rap1 upon cellular stimulation. Rasa3 is critical to restrain CD-GEFI/Rap1 signaling in circulating platelets; during hemostatic plug formation, however, its activity is downregulated after engagement of the platelet ADP receptor, P2Y12, the target of antiplatelet therapy. Mice lacking functional Rasa3 exhibit severe thrombocytopenia, caused by impaired production and premature clearance of platelets. Based on these and other studies we concluded that both platelet homeostasis and vascular hemostasis depend on a tight regulation of Rap signaling, and that a better understanding of these fundamental processes may have important implications in the diagnosis and treatment of disorders that affect platelet number and function. Utilizing unique mouse models, primary and immortilized MKs, and clinically relevant human platelet samples, we will study key questions concerning Rap1 signaling in megakaryocytes and platelets: how does a shift in the antagonistic balance between CD-GEFI and Rasa3 affect platelet survival? What is the role of Rap1 signaling in megakaryocyte development and platelet production? Are their different pools of Rap1 protein that regulate specific cellular responses in MKs and platelets? How similar are mouse and human platelets with regard to Rap1 signaling? What are the molecular mechanisms controlling Rasa3 activity downstream of P2Y12? To test the clinical relevance of our findings, we will investigate if increased Rap1 signaling in platelets and MKs, induced by impaired calcium homeostasis, is the underlying cause of the marked thrombocytopenia observed in Stormorken syndrome, and we will determine whether interindividual variability in the Rap1 signaling pathway contributes to P2Y12 inhibitor resistance in healthy individuals and patients with type 2 diabetes. If successful, these studies could pave the way to novel strategies for diagnosing and managing some of the inherited and acquired thrombocytopenias, and to a more personalized approach to anti-platelet therapy.